Herpesvirus saimiri (HVS) is a lymphotropic rhadinovirus (γ-2 herpesvirus) which causes persistent infection in its natural host the squirrel monkey (Saimiri sciureus) without causing any obvious symptoms of disease. HVS has been subdivided into three groups (A, B and C) on the basis of the sequence of the open reading frame of H. saimiri transformation-associated protein (STP) (Fleckenstein & Desrosiers, 1982; Medveczky et al., 1984). The structure of the HVS genome consists of a unique, low G+C content DNA segment (L-DNA) approximately 110 kb in length, flanked by multiple tandem repeats of high G+C content DNA (H-DNA) (Albrecht et al., 1992; Bankier et al., 1985). Analysis indicates it shares limited homology with other herpesviruses. Examples of such herpesviruses include Epstein Barr Virus (EBV), bovine herpesvirus 4 and murine gammaherpesvirus 68 (MHV68) (Blubot et al., 1992; 1996; Virgin et al., 1997). The genomes of EBV, BHV, MHV68 and HVS have been shown to be generally co-linear, in that homologous sequences are found in approximately equivalent locations and in the same relative orientation. However, conserved gene blocks are separated by unique genes with respect to each virus (Virgin et al., 1997). Genes which are expressed in HVS in the latent state are currently unknown.
HVS has a number of features which make it an attractive candidate for use as a gene delivery vector. These include the potential to package and deliver in excess of 50 kb of heterologous DNA, the ability to infect non-dividing cells and the maintenance of the viral genome as a stable episome in a latently infected host cell. The ability of herpes viruses to adopt a latent state in infected cells is a particularly attractive feature in terms of their use as gene delivery vehicles. In addition, because HVS is a non-human pathogen, it should not elicit a primary immune response on introduction into a human host. Primary immune response is a fundamental problem associated with human herpesvirus gene delivery systems which reduces the efficency of these vectors.
In our studies, we generated a recombinant HVS based on the non-transforming strain A11, which expresses the green fluorescent protein (GFP) gene (Whitehouse et al., 1998b). This virus contains the GFP gene under the control of the constitutive human cytomegalovirus (HCMV) early promoter inserted into the rightmost flanking region of H-DNA. We have demonstrated that this recombinant HVS-GFP was able to infect a wide range of human cancer cell lines, including T-cell (Jurkat), pancreatic (MIAPACA), colorectal (SW480) and lung carcinoma cells (A549). Thus, we have continued investigation of this recombinant HVS as we believe it to be an ideal candidate as a gene delivery vector.
The use of an efficient promoter which can drive stable long term expression of a transgene is a prerequisite for the development of any gene delivery vector. A variety of promoters have been utilised in herpes simplex virus (HSV) vectors including neuronal-specific promoters such as the neurone-specific enolase promoter, the neurofilament promoter and tyrosine hydroxylase promoter, as well as viral promoters such as the HSV thymidine kinase promoter and the HCMV immediate early promoter. Studies showed, however, that these promoters are unsuitable for long term expression in vivo, due to promoter silencing effects (Fink et al., 1996; Glorioso et al., 1992; 1995). There is a need, therefore, to identify viral regulatory regions which can be used to drive stable long term expression of a transgene.
Recently, recombinant HSV-1 viruses have been produced in which expression of the lacZ and lacZ-neo cassettes are driven by the latency-associated-transcript (LAT) promoter (Lachmann & Efstathiou, 1997). Peripheral infection of neurones with these viruses results in stable long-term expression of a β-galactosidase transgene for at least 190 days post-infection. Therefore, we believe that it would be advantageous to identify and characterise HVS regulatory regions associated with latency, if they exist, to drive long term stable expression of heterologous transgenes for the future development of HVS as a gene delivery system. In the course of our investigations to identify viral regulatory regions which can be used to drive stable long term expression of a transgene, we serendipitously identified a cluster of HVS genes which are apparently expressed specifically in the latent state and we provide evidence to this effect. The DNA sequence which unexpectedly drives expression of this series of transcripts has been identified. This sequence provides the advantages as a promoter to drive therapeutic gene expression discussed above.
In this application, we describe the identification of a cluster of genes encoding ORF71–73 which are latently expressed in an A549 cell line stably transduced by HVS-GFP. We have characterised a region of 2000 bp immediately upstream of the coding sequence of ORF73 and demonstrated that this regulatory region, when transfected into a human 293T cell line, is able to drive active expression of the GFP reporter gene. This result demonstrates that the upstream region of ORF73 contains regulatory sequences which may be utilized to drive expression of heterologous transgenes in a range of human cell lines. Therefore we believe that the ORF73 promoter, which drives virus-encoded gene expression whilst the HSV is present in a cell in a latent state, is an ideal choice of regulatory sequence for driving stable long term expression of a transgene in HVS-based gene delivery vectors.
Furthermore, in order to further investigate the possibility of using the ORF73 regulatory region as a promoter to drive long term expression of a heterologous transgene, a number of PCR fragments containing sequence immediately upstream of the ORF73 initiation codon were amplified by PCR and cloned into a reporter plasmid containing the GFP gene. These reporter constructs were transfected into the human 293T cell line and we have demonstrated that some of these fragments contain a regulatory region sufficient to drive heterologous gene expression in a human 293T cell line.
We believe that Herpesvirus saimiri (HVS) is an attractive candidate for use as a gene therapy vector as it has the ability to enter a latent mode of infection in which the viral genome is maintained as a stable episome in the host cell. We have generated a recombinant HVS in which the gene encoding green fluorescent protein (GFP) is expressed under the control of the constitutive human cytomegalovirus (CMV) promoter (HVS-GFP). This recombinant virus is able to stably transduce a range of human cell lines including the lung carcinoma cell line, A549, and direct production of GFP. However, it is known that the human CMV promoter is not effective in many circumstances for sustaining transgene expression in gene therapy in vivo. We have therefore sought to identify promoters which might be functional during latent infection with the HVS vectors.
Statement of the Invention
In the broadest aspect of the invention there is provided a gene delivery system/vaccine comprising a promoter which functions in a vector gene delivery system/vaccine during periods when the gene therapy vector is present in the cell in a latent state. The present invention is capable of regulating long term gene expression in the gene delivery system/vaccine and is capable of controling the expression of transgenes in a range of human or animal cells.
According to a first aspect of the invention there is provided a nucleic acid comprising a nucleic acid sequence which encodes a promoter and which hybridises under high stringency conditions to the nucleic acid sequence of SEQ ID NO:1, fragments and/or variants thereof, for use in gene therapy.
Preferably, hybridisation occurs under stringent conditions such as 1× SSC, 0.1% SDS at 65° C.
Preferably, said promoter comprises a nucleic acid sequence of at least 329 bp and up to 2000 bp, more preferably said promoter comprises a nucleic acid sequence of up to a length of 329, 630, 1000 or 1500 bp or any other selected fragment or variant thereof. It will be appreciated that it is possible that the promoter sequence of the invention may be less than 329 bp so long as the effective sequence encoding the promoter is included in the invention.
According to a second aspect of the invention there is provided a recombinant DNA molecule containing at least one insert comprising the nucleic acid sequence of SEQ ID NO:1, fragment or variant thereof, encoding a promoter.
Thus it will be appreciated that the invention includes nucleic acids comprising (i) a sequence of up to 2000 bp which encodes the promoter, (ii) fragments of selected bp lengths within the sequence and (iii) variants thereof, as well as recombinant DNA molecules containing insert(s) of the promoter sequence therein.
According to a third aspect of the invention there is provided a gene therapy system comprising a vector which includes a nucleic acid sequence selected from the group consisting of the nucleic acid sequence of SEQ ID NO:1, and fragments and variants thereof as well as nucleic acid sequences which hybridise under high stringency conditions to the sequence of SEQ ID NO:1, or a part thereof, wherein said system is capable of driving heterologous gene expression during periods of latent infection by the vector in a target cell population.
Preferably, the gene therapy system further includes any one or more of the features herein before described.
Preferably, said vector additionally comprises at least one therapeutic nucleic acid, whereby the promoter encoded by SEQ ID NO:1 or fragment or variant thereof acts to drive expression of said the at least one therapeutic nucleic acid.
Reference herein to therapeutic nucleic acid is intended to include a therapeutic gene or fragment or variant thereof.
The vector of said gene delivery system may be viral or non-viral.
Preferably, said gene therapy system is capable of long term gene expression.
Reference herein to long term gene expression includes gene expression for at least several hours and optimally at least several months, for example and without limitation, from 2 hours to six months or more.
According to a fourth aspect of the invention there is provided use of a gene therapy system as herein before described for long term gene expression.
It will be appreciated by those skilled in the art that the invention comprises a gene therapy system and that, in preferred embodiments the vector may be either viral or non-viral. The expression of a therapeutic gene can be regulated by a promoter, typically of up at least 329 bp and up to 2000 bp, the system being capable of driving heterologous gene expression during periods of latent infection of a target cell population. Thus, foreign transgenes can be controlled by, for example, a natural promoter, which is active in the latent mode of viral infection. The specifics of the gene expression and the nature of the vector is not intended to limit the scope of the application.
According to a fifth aspect of the invention there is provided an HVS comprising a nucleic acid sequence encoding a promoter of SEQ ID NO:1, or fragment or variant thereof or a nucleic acid sequence which hybridises under high stringency conditions to the sequence of SEQ ID NO:1, fragment or variant thereof, which promoter acts in the latent state, the sequence encoding for the promoter being positioned so as to drive expression of at least one therapeutic nucleic acid which has been inserted in the HVS.
The preferred embodiments of the fifth aspect of the invention include those listed in accordance with the aforementioned first and third aspects of the invention.
Preferably, the HVS of the present invention may be rendered ineffective and its activity terminated by the appropriate co-administration of an anti-herpetic pharmaceutical such as acyclovir.
According to a sixth aspect of the invention there is provided a method of manufacturing an expression vector comprising the promoter of the first aspect of the invention or the gene therapy system of the third aspect of the invention or the HVS vector of the fifth aspect of the invention, the method comprising transfecting a cell with a nucleic acid sequence encoding said promoter of SEQ ID NO:1, or fragment or variant thereof or a nucleic acid sequence which hybridises under high stringency conditions to the sequence of SEQ ID NO:1 or any part thereof.
The invention includes methods which comprise selecting the promoter and amplifying it and subsequently purifying it prior to transfecting a cell population, preferably a selected target cell population.
According to a seventh aspect of the invention there is provided a method of treatment comprising administering a therapeutically effective amount of the promoter of the first aspect of the invention or a gene therapy system of the third aspect of the invention or a HVS gene therapy vector of the fifth aspect of the invention, to an individual requiring treatment.
According to an eighth aspect there is provided the promoter of the first aspect of the invention or the gene therapy system of the third aspect of the invention or the HVS vector of the fifth aspect of the invention for use as a pharmaceutical.
According to a yet further aspect of the invention there is provided a pharmaceutical composition comprising the promoter of the first aspect of the invention or a gene therapy system of the third aspect of the invention or a HVS gene therapy vector of the fifth aspect of the invention, the pharmaceutical additionally comprises a pharmaceutically acceptable excipient, diluent or carrier and ideally said pharmaceutical can be formulated as a nasal spray, or for injection or for oral/paraenteral administration into a individual requiring treatment.
According to a yet further aspect of the invention there is provided use of the promoter of the first aspect of the invention or the gene therapy system of the third aspect of the invention or the HVS vector of the fifth aspect of the invention in the manufacture of a medicament for treating cancer.
According to a yet further aspect of the invention there is provided use of the promoter of the first aspect of the invention or the gene therapy system of the third aspect of the invention or the HVS vector of the fifth aspect of the invention in the manufacture of a medicament for treating degenerative disorders.
According to a yet further aspect of the present invention is an isolated nucleic acid encoding a promoter, the nucleic acid may be selected from the group consisting of:                (a) DNA having the nucleotide sequence given herein as SEQ ID NO:1 and which encodes the promoter;        (b) nucleic acids which hybridize to DNA of (a) above (e.g., under stringent conditions) and which encode the promoter; andDNAs of the present invention include those of closely related sequences to, and having essentially the same biological properties as, the promoter disclosed herein, and particularly the DNA disclosed herein as SEQ ID NO:1. This definition is intended to encompass natural allelic variations therein. Thus, DNAs which hybridize to DNA disclosed herein as SEQ ID NO:1 (or fragments or derivatives thereof which serve as hybridization probes as discussed below) and which encode the promoter of the present invention are to be included in the definition.        
Conditions which will permit other DNAs which encode the promoter of the present invention and hybridize to the DNA of SEQ ID NO:1 disclosed herein can be determined in accordance with known techniques. For example, hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35–40% Formamide with 5× Denhardt's solution, 0.5% SDS and 1× SSPE at 37° C.; conditions represented by a wash stringency of 40–45% Formamide with 5× Denhardt's solution, 0.5% SDS, and 1× SSPE at 42° C.; and conditions represented by a wash stringency of 50% Formamide with 5× Denhardt's solution, 0.5% SDS and 1× SSPE at 42° C., respectively) to DNA of SEQ ID NO:1 disclosed herein in a standard hybridization assay. See, e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory). In general, sequences which code the promoter of the present invention and which hybridize to the DNA of SEQ ID NO:1 disclosed herein will be at least 75% homologous, 85% homologous, and even 95% homologous or more with SEQ ID NO:1.